I. Field of the Invention
Embodiments of this invention are directed generally to biology, medicine, and diagnostics. In particular aspects of the invention are directed to detection of single molecule detection using two-dimensional photon counting analysis.
II. Background
Single molecule detection (SMD) with the assistance of micro/nano-fluidic devices has attracted tremendous amount of attention [1]. It provides a powerful way of biomolecular detection compare with conventional biosensors. Most conventional bio-analytical sensors utilize ensemble measurements and only yield information on the average for the entire population in a certain time frame. However, they seldom deal with heterogeneous samples, therefore, any fluctuation, reaction intermediate states, and time trajectories will affect the accuracy of detection in the conventional ensemble measurements [2]. SMD techniques, on the other hand, are able to provide us with invaluable information of molecular dynamics in many aspects that would otherwise be hidden and sometimes impossible to obtain with conventional techniques [3]. Micro/nano-fluidic technology has also developed rapidly over the last ten years [4-9]. It offers a spatial confinement of molecules in one or two dimensions in a continuous flow system. This will not only ensure a fixed position for interrogation of target molecules but also avoid repeated detection of the same molecule. As channel dimensions shrink and become comparable or smaller than the optical excitation volume, uniform excitation of target molecules and very high detection efficiency can be achieved. In addition, signal-to-noise ratio is improved significantly as the background from scattering and/or intrinsic fluorescence of unlabelled species in the probe volume is minimized. Sometimes SMD can be difficult to achieve while trying to investigate molecules in their native environment or at their physiological concentrations. With the help of micro/nano-fluidic devices, this has become feasible. The implementation of miniaturized devices greatly reduces sample consumption and as lab-on-a-chip technology advances, integrated high-throughput parallel detection system becomes feasible in the near future. By merging these two techniques, it is obvious that the inventors can achieve the optimal requirements for the analysis and manipulation of samples on a single molecule level [1, 10-12] and it had already found applications in many different fields, such as DNA separation [13-15], sequencing [16], mapping and fragment sizing [17-22], molecular conformation studies [23, 24], drug screening, chemical analysis [25,26], microflow characterization [27], and ultra-sensitive detection without target amplification [28].
Besides the aforementioned fields, molecule-molecule interaction studies at single molecule level in bulk solutions, on planer surfaces [29-35], and in microfluidic flowing environment [36-40] have become an active research area in recent years. Stavis and co-workers demonstrated efficient multicolor fluorescence detection and characterization of QD655 Streptavidin Conjugates binding to Alexa Fluor 488 molecules in a submicrometer fluidic channel [36]. Zhang et al. introduced a homogenous technique for rapid and sensitive probing specific DNA molecules using two-color quantum dots based on single-molecule coincidence detection in a capillary with inner diameter of 50 μm [40]. Most recently, Agrawal and co-workers reported the use of bioconjugated nanoparticles and two-color fluorescence coincidence for real-time detection of purified single gene, protein and intact virus in a flowing fused silica capillary with inner diameter of 2 μm [38]. Two-color channel detection is one of the common schemes in fluorescence-based molecule-molecule interaction studies at single molecule level. This requires two separated optical paths and detectors in the system. Spectral cross-talk still poses potential problems for two-color schemes but the use of quantum dots (QDs) with narrow emission bandwidth can alleviate the problem. However, very few specific protein detection studies are available currently in the literature due to difficulties in handling protein molecules in a fused silica microfluidic channel. There remains a need for additional methods for single molecule detection.